CN102939376B - Novel cellulase gene - Google Patents

Abstract

The object of the present invention is to isolate the genomic DNA containing the cellulase gene classified into endoglucanase and β-glucosidase from Acremonium cellulolyticum, and determine the endoglucanase gene by analyzing its base sequence. Glycanase and β-glucosidase genes. The amino acid sequences of known endoglucanases and β-glucosidases were thoroughly compared, and amino acid sequences that were also conserved in Acremonium cellulolyticum were found, and various primers were designed based on this information. Using various primers thus designed, PCR was carried out using genomic DNA or cDNA as a template. As a result, gene fragments of endoglucanase and β-glucosidase were obtained. Therefore, based on this gene fragment, primers were designed, and 9 kinds of endoglucanase and β-glucanase were amplified by continuing PCR. -The gene of glucosidase, and its base sequence was analyzed, thereby completing the present invention.

Description

new cellulase gene

technical field

The present invention relates to cellulase, in particular to cellulase derived from Acremonium cellulolyticus, a polynucleotide encoding the cellulase, a method for producing cellulase using the polynucleotide, and uses thereof . Furthermore, in the present specification, "polynucleotide" includes, for example, DNA or RNA, or their modified or chimera, preferably DNA.

Background technique

Cellulase is a general term for enzymes that decompose cellulose. Generally speaking, cellulase produced by microorganisms includes a variety of cellulase components. Cellulase components can be divided into three types according to their substrate specificity: cellobiohydrolase, endoglucanase, and β-glucosidase. ), it can be considered that it produces up to 4 cellobiohydrolases, 15 endoglucanases, and 15 β-glucosidases. Therefore, when the cellulase produced by microorganisms is used industrially, it is used as a mixture of various cellulase components produced by the microorganisms.

The filamentous fungus Acremonium cellulolyticum is characterized by the production of cellulase with strong saccharifying power (Non-Patent Document 1), and it has been reported that it is highly useful in feed and silage applications (Patent Documents 1-3 ). Furthermore, the cellulase components contained therein have been studied in detail (Patent Documents 4-10), and it is known that they secrete various cellulase components like other filamentous fungi.

It is considered that, when limited to a certain application, specific several enzyme components are important for the application among the plurality of cellulase components. Therefore, if the composition of cellulase components can be optimized for cellulase produced by microorganisms according to the usage, it can be expected to obtain cellulase with higher activity. The best method so far is to introduce a specific enzyme gene for overexpression or deletion of a specific enzyme gene by gene recombination.

However, in the case of Acremonium cellulolyticum, only two cellobiohydrolase genes (Patent Documents 4 and 5) and one β-glucosidase gene ( Patent Document 10), the current situation is that, for other cellulases, expression enhancement by gene introduction or expression suppression by deletion cannot be performed.

Against such a background, in order to optimize the composition of cellulase produced by Acremonium cellulolyticum by genetic recombination technology, it is desired to isolate polysaccharide decomposing enzyme genes such as endoglucanase and β-glucosidase.

prior art literature

non-patent literature

Non-Patent Document 1: Agricultural and Biological Chemistry, (Japan), 1987, Vol. 51, p.65

patent documents

Patent Document 1: Japanese Patent Application Laid-Open No. 7-264994

Patent Document 2: Specification of Japanese Patent No. 2531595

Patent Document 3: Japanese Patent Application Laid-Open No. 7-236431

Patent Document 4: Japanese Patent Laid-Open No. 2001-17180

Patent Document 5: International Publication No. WO97/33982 Pamphlet

Patent Document 6: International Publication WO99/011767 Pamphlet

Patent Document 7: Japanese Patent Laid-Open No. 2000-69978

Patent Document 8: Japanese Patent Application Laid-Open No. 10-066569

Patent Document 9: Japanese Patent Laid-Open No. 2002-101876

Patent Document 10: Japanese Patent Laid-Open No. 2000-298262

Contents of the invention

The problem to be solved by the invention

The subject of the present invention is to determine the endonuclease gene by isolating the genome DNA containing the cellulase gene classified into endoglucanase and β-glucosidase from Acremonium cellulolyticum and analyzing its base sequence. Glucanase and β-glucosidase genes.

way of solving the problem

In order to solve the above problems, the present invention made an in-depth comparison of the amino acid sequences of known endoglucanases and β-glucosidases, found a conserved amino acid sequence even in Acremonium cellulolyticum, and based on Various primers were designed for this information. Using various primers thus designed, PCR was carried out using genomic DNA or cDNA as a template. As a result, the gene fragments of endoglucanase and β-glucosidase were obtained, so primers were designed based on this gene fragment, and PCR was continued to amplify 9 kinds of endoglucanase and β-glucosidase Enzyme gene, and carried out base sequence analysis, thereby completing the present invention.

That is, the present invention relates to:

[1] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 306 of the amino acid sequence described in SEQ ID NO: 2;

(ii) an endoglucanase comprising an amino acid sequence obtained by deleting, substituting, and/or adding one or more amino acids in the sequence at positions 1 to 306 of the amino acid sequence described in SEQ ID NO: 2;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 306 of the amino acid sequence described in SEQ ID NO: 2,

[2] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 475 of the amino acid sequence described in SEQ ID NO: 4;

(ii) an endoglucanase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 475 of the amino acid sequence described in SEQ ID NO: 4;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 475 of the amino acid sequence described in SEQ ID NO: 4,

[3] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 391 of the amino acid sequence described in SEQ ID NO: 6;

(ii) an endoglucanase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 391 of the amino acid sequence described in SEQ ID NO: 6;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 391 of the amino acid sequence described in SEQ ID NO: 6,

[4] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 376 of the amino acid sequence described in SEQ ID NO: 8;

(ii) an endoglucanase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 376 of the amino acid sequence described in SEQ ID NO: 8;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 376 of the amino acid sequence described in SEQ ID NO: 8,

[5] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 221 of the amino acid sequence described in SEQ ID NO: 10;

(ii) an endoglucanase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 221 of the amino acid sequence described in SEQ ID NO: 10;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 221 of the amino acid sequence described in SEQ ID NO: 10,

[6] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 319 of the amino acid sequence described in SEQ ID NO: 12;

(ii) an endoglucanase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 319 of the amino acid sequence described in SEQ ID NO: 12;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 319 of the amino acid sequence described in SEQ ID NO: 12,

[7] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence at positions 1 to 301 of the amino acid sequence described in SEQ ID NO: 14;

(ii) an endoglucanase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 301 of the amino acid sequence described in SEQ ID NO: 14;

(iii) an endoglucanase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 301 of the amino acid sequence described in SEQ ID NO: 14,

[8] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 458 of the amino acid sequence described in SEQ ID NO: 16;

(ii) β-glucosidase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 458 of the amino acid sequence described in SEQ ID NO: 16;

(iii) a β-glucosidase comprising an amino acid sequence having 70% or more identity to the sequence at positions 1 to 458 of the amino acid sequence described in SEQ ID NO: 16,

[9] A protein selected from (i), (ii) and (iii) below:

(i) a protein comprising the sequence of positions 1 to 457 of the amino acid sequence described in SEQ ID NO: 18;

(ii) β-glucosidase comprising an amino acid sequence obtained by deletion, substitution, and/or addition of one or more amino acids in the sequence at positions 1 to 457 of the amino acid sequence described in SEQ ID NO: 18;

(iii) a β-glucosidase comprising an amino acid sequence having 70% or more identity with the sequence at positions 1 to 457 of the amino acid sequence described in SEQ ID NO: 18,

[10] The protein described in [1] to [9] derived from a filamentous fungus,

[11] The protein according to [10], wherein the filamentous fungus is a filamentous fungus belonging to Acremonium cellulolyticus,

[12] A polynucleotide comprising a base sequence encoding the protein described in [1] to [9],

[13] DNA comprising the nucleotide sequence described in SEQ ID NO: 1 or a modified sequence thereof,

[14] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [1];

(ii) DNA comprising the sequence at positions 136 to 1437 of the base sequence described in SEQ ID NO: 1;

(iii) DNA hybridized under stringent conditions with the sequence consisting of 136 to 1437 positions of the base sequence described in SEQ ID NO: 1, and encoding a protein with endoglucanase activity,

[15] DNA obtained by removing an intron sequence from the DNA described in [14],

[16] The DNA described in [15], wherein the intron sequence is selected from 233~291, 351~425, 579~631, 697~754 of the base sequence described in SEQ ID NO: 1 or a sequence of one or more of the sequences of 853 to 907 positions,

[17] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [13] to [16],

[18] The DNA described in [17], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 136 to 216 of the nucleotide sequence described in SEQ ID NO: 1,

[19] DNA comprising the nucleotide sequence described in SEQ ID NO: 3 or a modified sequence thereof,

[20] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [2];

(ii) DNA comprising the sequence at positions 128 to 1615 of the base sequence described in SEQ ID NO: 3;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of 128 to 1615 of the base sequence described in SEQ ID NO: 3 and encodes a protein with endoglucanase activity,

[21] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [19] to [20],

[22] The DNA described in [21], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 128 to 187 of the nucleotide sequence described in SEQ ID NO: 3,

[23] DNA comprising the nucleotide sequence described in SEQ ID NO: 5 or a modified sequence thereof,

[24] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [3];

(ii) DNA comprising the sequence of 169 to 1598 of the base sequence described in SEQ ID NO: 5;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of DNA at positions 169 to 1598 of the base sequence described in SEQ ID NO: 5, and encodes a protein with endoglucanase activity,

[25] DNA obtained by removing an intron sequence from the DNA described in [24],

[26] The DNA described in [25], wherein the intron sequence is selected from the sequence consisting of 254-309, 406-461 or 1372-1450 of the base sequence described in SEQ ID NO: 5 More than one sequence sequence,

[27] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [23] to [26],

[28] The DNA described in [27], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 169 to 231 of the nucleotide sequence described in SEQ ID NO: 5,

[29] DNA comprising the nucleotide sequence described in SEQ ID NO: 7 or a modified sequence thereof,

[30] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [4];

(ii) DNA comprising the sequence of 70-1376 of the base sequence described in SEQ ID NO: 7;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of DNA at positions 70 to 1376 of the base sequence described in SEQ ID NO: 7, and encodes a protein with endoglucanase activity,

[31] DNA obtained by removing an intron sequence from the DNA described in [30],

[32] The DNA described in [31], wherein the intron sequence is a sequence of one or more sequences selected from the sequence at positions 451 to 500 or 765 to 830 of the base sequence described in SEQ ID NO: 7 ,

[33] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [29] to [32],

[34] The DNA described in [33], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 70 to 129 of the nucleotide sequence described in SEQ ID NO: 7,

[35] DNA comprising the nucleotide sequence described in SEQ ID NO: 9 or a modified sequence thereof,

[36] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [5];

(ii) DNA comprising the sequence at positions 141 to 974 of the base sequence described in SEQ ID NO: 9;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of 141 to 974 positions of the base sequence described in SEQ ID NO: 9, and encodes a protein with endoglucanase activity,

[37] DNA obtained by removing an intron sequence from the DNA described in [36],

[38] The DNA described in [37], wherein the intron sequence comprises one or more sequences selected from the sequence at positions 551 to 609 or 831 to 894 of the base sequence described in SEQ ID NO: 9 sequence,

[39] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [35] to [38],

[40] The DNA described in [39], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 141 to 185 of the nucleotide sequence described in SEQ ID NO: 9,

[41] DNA comprising the nucleotide sequence described in SEQ ID NO: 11 or a modified sequence thereof,

[42] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [6];

(ii) DNA comprising the sequence of 114 to 1230 of the base sequence described in SEQ ID NO: 11;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of 114 to 1230 of the base sequence described in SEQ ID NO: 11, and encodes a protein with endoglucanase activity,

[43] DNA obtained by removing an intron sequence from the DNA described in [42],

[44] The DNA described in [43], wherein the intron sequence comprises one or more sequences selected from the sequence at positions 183-232 or 299-357 of the base sequence described in SEQ ID NO: 11 sequence,

[45] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [41] to [44],

[46] The DNA described in [45], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 114 to 161 of the nucleotide sequence described in SEQ ID NO: 11,

[47] DNA comprising the nucleotide sequence described in SEQ ID NO: 13 or a modified sequence thereof,

[48] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [7];

(ii) DNA comprising the sequence at positions 124 to 1143 of the base sequence described in SEQ ID NO: 13;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of 124 to 1143 of the base sequence described in SEQ ID NO: 13, and encodes a protein with endoglucanase activity,

[49] DNA obtained by removing an intron sequence from the DNA described in [48],

[50] The DNA described in [49], wherein the intron sequence is a sequence at positions 225 to 275 of the base sequence described in SEQ ID NO: 13,

[51] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [47] to [50],

[52] The DNA described in [51], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 124 to 186 of the nucleotide sequence described in SEQ ID NO: 13,

[53] DNA comprising the nucleotide sequence described in SEQ ID NO: 15 or a modified sequence thereof,

[54] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [8];

(ii) DNA comprising the sequence at positions 238 to 1887 of the base sequence described in SEQ ID NO: 15;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of DNA at positions 238 to 1887 of the base sequence described in SEQ ID NO: 15, and encodes a protein with β-glucosidase activity,

[55] DNA obtained by removing an intron sequence from the DNA described in [54],

[56] The DNA described in [55], wherein the intron sequence is 1 selected from the sequence of 784-850, 1138-1205 or 1703-1756 of the base sequence described in SEQ ID NO: 15 More than one sequence sequence,

[57] DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [53] to [56],

[58] The DNA described in [57], wherein the nucleotide sequence encoding the signal sequence is a sequence at positions 238 to 321 of the nucleotide sequence described in SEQ ID NO: 15,

[59] DNA comprising the nucleotide sequence described in SEQ ID NO: 17 or a modified sequence thereof,

[60] A DNA selected from (i), (ii) and (iii) below:

(i) DNA encoding the protein described in [9];

(ii) DNA comprising the sequence at positions 66 to 1765 of the base sequence described in SEQ ID NO: 17;

(iii) a DNA that hybridizes under stringent conditions with the sequence consisting of DNA at positions 66 to 1765 of the base sequence described in SEQ ID NO: 17, and encodes a protein with β-glucosidase activity,

[61] DNA obtained by removing an intron sequence from the DNA described in [60],

[62] The DNA described in [61], wherein the intron sequence is selected from 149~211, 404~460, 934~988 or 1575~1626 of the base sequence described in SEQ ID NO: 17 A sequence of more than one sequence in the sequence,

[63] The DNA obtained by removing the nucleotide sequence encoding the signal sequence from the DNA described in [59] to [62],

[64] The DNA described in [63], the nucleotide sequence encoding the signal sequence is the sequence at positions 66 to 227 of the nucleotide sequence described in SEQ ID NO: 17,

[65] An expression vector comprising the DNA described in [12] to [64],

[66] A host cell transformed with the expression vector described in [65],

[67] The host cell of [66], wherein the host cell is a yeast or a filamentous fungus,

[68] The host cell described in [67], wherein the yeast belongs to the genus Saccharomyces , Hansenula or Pichia ,

[69] The host cell of [68], wherein the yeast is Saccharomyces cerevisiae ,

[70] The host cell of [67], wherein the filamentous fungus belongs to the genus Humicola , Aspergillus , Trichoderma , Fusarium or Acremonium Genus ( Acremonium ),

[71] The host cell described in [70], wherein the filamentous fungus is Acremonium cellulolyticus, Humicola insolens , Aspergillus niger or Aspergillus oryzae, Viridian wood Mold ( Trichodermaviride ), or Fusarium oxysporum ( Fusarium oxysporum ),

[72] A filamentous fungus of the genus Acremonium in which a gene corresponding to the DNA described in [12] to [64] has been deleted by homologous recombination,

[73] The filamentous fungus of [72], wherein the filamentous fungus is Acremonium cellulolyticus,

[74] The method for producing the protein described in [1] to [9], which comprises culturing the host cell described in [66] to [73], and collecting [1] to [ from the host and/or its culture 9] the steps of the protein,

[75] A protein produced by the method described in [74],

[76] A cellulase preparation comprising the protein described in [1] to [9] or [75],

[77] A method for saccharifying biomass, the method comprising: mixing biomass containing cellulose with the protein described in [1] to [9] or [75], or the fiber described in [76] The step of contacting the prime enzyme preparation,

[78] A method for treating cellulose-containing fibers, the method comprising: mixing cellulose-containing fibers with the protein described in [1] to [9] or [75], or the cellulose described in [76] The step of contacting the enzyme preparation,

[79] A method for deinking waste paper, characterized in that, in the step of treating waste paper with a deinking agent for deinking, using the protein described in [1] to [9] or [75], or [ 76] the cellulase preparation,

[80] A method for improving the drainage of paper or pulp, the method comprising: using the protein described in [1] to [9] or [75], or the cellulase described in [76] with paper or pulp the steps in which the preparation is processed,

[81] A method for improving the digestibility of animal feed, the method comprising subjecting animal feed to the protein described in [1] to [9] or [75], or the cellulase preparation described in [76] processing steps.

The effect of the invention

According to the present invention, the DNA necessary for efficient production of specific endoglucanase and β-glucosidase derived from Acremonium cellulolyticum in the form of recombinant protein can be obtained, and in addition, high-efficiency expression of these cellulases can be obtained Components of recombinant microorganisms. Furthermore, specific endoglucanase and β-glucosidase can be efficiently and inexpensively produced by culturing the obtained recombinant microorganism.

In addition, according to the present invention, specific endoglucanase and β-glucosidase genes can be deleted in the genome of Acremonium cellulolyticum, and as a result, it is possible to obtain products that do not contain these endoglucanases and β-glucosidase cellulase recombinant Acremonium cellulolyticum can produce cellulase free of specific endoglucanase and β-glucosidase cellulase.

Among various cellulase enzymes obtained by the present invention, by selecting an optimal group of cellulase enzymes and treating cellulosic substrates, these cellulosic substrates can be efficiently and inexpensively decomposed.

Description of drawings

[Fig. 1] Restriction enzyme map of plasmid pACC3.

[Fig. 2] Restriction enzyme map of plasmid pACC5.

[Fig. 3] Restriction enzyme map of plasmid pACC6.

[Fig. 4] Restriction enzyme map of plasmid pACC7.

[Fig. 5] Restriction enzyme map of plasmid pACC8.

[Fig. 6] Restriction enzyme map of plasmid pACC9.

[ Fig. 7 ] Restriction enzyme map of plasmid pACC10.

[Fig. 8] Restriction enzyme map of plasmid pBGLC.

[Fig. 9] Restriction enzyme map of plasmid pBGLD.

Specific Embodiments of the Invention

Endoglucanase and β-glucosidase

Endoglucanase and β-glucosidase as the protein of the present invention may comprise a mature The sequence of the protein moiety may alternatively comprise an amino acid sequence substantially identical to the amino acid sequence.

In the present invention, "substantially identical amino acid sequence" refers to: for example, having one or more (preferably several) amino acid substitutions, deletions, and/or modifications caused by additions, but do not affect the activity of the polypeptide. amino acid sequence, or an amino acid sequence that has more than 70% identity but does not affect the activity of the polypeptide.

The number of amino acid residues to be modified is preferably 1 to 40, more preferably 1 to several, more preferably 1 to 8, and most preferably 1 to 4. Examples of "modifications that do not affect activity" in the present invention include conservative substitutions. "Conservative substitution" refers to the substitution of one or more amino acid residues with other chemically similar amino acid residues without substantially changing the activity of the polypeptide. Examples thereof include the case of substituting a certain hydrophobic residue with another hydrophobic residue, and the case of substituting a certain polar residue with another polar residue having the same charge. Functionally similar amino acids capable of such substitutions are well known in the art by amino acid classification. Specific examples include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine as nonpolar (hydrophobic) amino acids. Wait. Glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine etc. are mentioned as a polar (neutral) amino acid. Arginine, histidine, lysine etc. are mentioned as an amino acid which has a positive charge (basic). Moreover, aspartic acid, glutamic acid, etc. are mentioned as an amino acid which has a negative charge (acidity).

"Identity (identity)" in this specification means: FASTA3 [Science, 227, 1435-1441 (1985); Proc.Natl.Acad.Sci.USA, which is a homology search program known to those skilled in the art , 85, 2444-2448 (1988); http://www.ddbj.nig.ac.jp/E-mail/homology-j.html], the value calculated using the default (initial setting) parameters. The identity may be preferably 80% or more identity, more preferably 90% or more identity, more preferably 95% or more identity, more preferably 98% or more identity, particularly preferably 99% or more identity sex.

To the protein of the present invention, any polypeptide sequence can be imparted to the N-terminal and/or C-terminal of each amino acid sequence corresponding to the mature protein portion, or an amino acid sequence substantially equivalent thereto, within a range that does not affect the enzymatic activity. Such a polypeptide sequence includes, for example, a signal sequence, a label for detection (eg, FLAG tag), and a polypeptide for purification [eg, glutathione sulfur transferase (GST)].

endoglucanase and β-glucosidase genes

The endoglucanase and β-glucosidase genes as polynucleotides of the present invention may comprise the base sequence encoding the protein of the present invention, and may also comprise bases selected from SEQ ID NO: 1. The sequence of 136-1437 positions of the base sequence, the sequence of 128-1615 positions of the base sequence described in SEQ ID NO: 3, the sequence of 169-1598 positions of the base sequence described in SEQ ID NO: 5, the sequence of positions 169-1598 of the base sequence described in SEQ ID NO: 7 The sequence at positions 70 to 1376 of the base sequence, the sequence at positions 141 to 974 of the base sequence described in SEQ ID NO: 9, the sequence at positions 114 to 1230 of the base sequence described in SEQ ID NO: 11, the sequence at positions 114 to 1230 of the base sequence described in SEQ ID NO: 13 The sequence of 124-1143 of the nucleotide sequence, the sequence of 238-1887 of the nucleotide sequence described in SEQ ID NO: 15, the nucleotide sequence of the 66-1765 sequence of the nucleotide sequence described in SEQ ID NO: 17 , or may contain a nucleotide sequence that can hybridize to the nucleotide sequence under stringent conditions.

In the present invention, "stringent conditions" refers to the washing operation of the hybridized membrane at high temperature and in a solution with low salt concentration, for example: 2×SSC concentration (1×SSC: 15mmol/L trisodium citrate , 150mmol/L sodium chloride), 0.5% SDS solution, 60 ° C, 20 minutes of washing conditions.

Acquisition of endoglucanase and β-glucosidase genes

The endoglucanase and β-glucosidase genes of the present invention can be isolated from Acremonium cellulolyticum or its mutant strains, for example, by the following method. In addition, as disclosed in the present invention, the base sequence is already known, so it can also be synthesized artificially.

Genomic DNA was extracted from Acremonium cellulolyticum cells by conventional methods. The genomic DNA was digested with an appropriate restriction enzyme and ligated with an appropriate vector to prepare a genomic DNA library of Acremonium cellulolyticum. As the vector, various vectors such as plasmid vectors, phage vectors, cosmid vectors, and BAC vectors can be used.

Then, appropriate probes can be made based on the base sequences of the endoglucanase and β-glucosidase genes disclosed in this specification, and the desired endoglucanase and β-glucanase genes can be isolated from the genomic DNA library by hybridization. A DNA fragment of the β-glucosidase gene. In addition, primers for amplifying desired genes can be prepared based on the nucleotide sequences of the endoglucanase and β-glucosidase genes disclosed in this specification, using the genomic DNA of Acremonium cellulolyticum as a template PCR is performed, and the desired gene is isolated by ligating the amplified DNA fragment with an appropriate vector. Moreover, the endoglucanase and β-glucosidase genes according to the present invention are contained in the plasmids pACC3, pACC5, pACC6, pACC7, pACC8, pACC9, pACC10, pBGLC and plasmid pBGLD, and thus these can be used as PCR substrates. Template DNA. In addition, desired DNA fragments can be prepared from these plasmids using appropriate restriction enzymes.

preservation of microorganisms

Escherichia coli transformed with pACC3 (Escherichia coli TOP10/pACC3) was deposited on October 9, 2008 at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (Postal code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11029.

Escherichia coli transformed with pACC5 (Escherichia coli TOP10/pACC5) was deposited on October 9, 2008 at the Patent Organisms Depository Center of the National Institute of Advanced Industrial Science and Technology (Zip code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11030.

Escherichia coli transformed with pACC6 (Escherichia coli TOP10/pACC6) was deposited on October 9, 2008 at the Patent Organisms Depository Center of the National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11031.

Escherichia coli transformed with pACC7 (Escherichia coli TOP10/pACC7) was deposited on October 9, 2008 at the Patent Organisms Depository Center of the National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11032.

Escherichia coli transformed with pACC8 (Escherichia coli TOP10/pACC8) was deposited on October 9, Heisei 20 (2008) at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (Zip Code 305-8566, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11033.

Escherichia coli transformed with pACC9 (Escherichia coli TOP10/pACC9) was deposited on October 9, 2008 at the Patent Organisms Depository Center of the National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11034.

Escherichia coli transformed with pACC10 (Escherichia coli TOP10/pACC10) was deposited on October 9, 2008 at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11035.

Escherichia coli transformed with pBGLC (Escherichia coli TOP10/pBGLC) was deposited on October 9, 2008 at the Patent Organisms Depository Center of the National Institute of Advanced Industrial Science and Technology (Postal code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11036.

Escherichia coli transformed with pBGLD (Escherichia coli TOP10/pBGLD) was deposited on October 9, 2008 at the Patent Organisms Depository Center of the National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) 1 Fandi 1 Central No. 6) has been deposited internationally. The accession number is FERMBP-11037.

Expression vectors and transformed microorganisms

In the present invention, a kind of expression vector is provided, and this expression vector can be replicated in host microorganism, and the state that the protein encoded by its DNA sequence can express comprises the SEQIDNO:2,4,6,8, DNA of the amino acid sequence described in 10, 12, 14, 16, or 18, or the nucleotide sequence of a modified amino acid sequence thereof (hereinafter also referred to as "the DNA sequence according to the present invention"). The expression vector can basically be constructed as a self-replicating vector, that is, it exists in the form of an extrachromosomal independent body, and its replication does not depend on the replication of the chromosome, such as a plasmid. In addition, when the present expression vector is introduced into a host microorganism, it can be integrated into the genome of the host microorganism and replicated together with the chromosome into which it has been integrated. The procedures and methods of vector construction according to the present invention can adopt those routinely used in the field of genetic engineering.

As far as the expression vector according to the present invention is concerned, in order to actually introduce it into the host microorganism and express a protein with desired activity, in addition to comprising the DNA sequence according to the present invention, it preferably also contains a DNA sequence for controlling its expression, Gene markers for selection of microorganisms, etc. The DNA sequence controlling expression includes a promoter, a terminator, a DNA sequence encoding a signal peptide, and the like. The promoter is not particularly limited as long as it can exhibit transcriptional activity in the host microorganism, and it can be obtained as a DNA sequence that controls the expression of a gene encoding any protein of the same or different species as the host microorganism. In addition, the signal peptide is not particularly limited, as long as it can contribute to the secretion of the protein in the host microorganism, and it can be obtained from a DNA sequence derived from a gene encoding any protein of the same or different species as the host microorganism. In addition, the gene markers in the present invention can be appropriately selected depending on the selection method of transformants, and for example, genes encoding drug resistance and genes complementary to nutritional requirements can be used.

Furthermore, according to the present invention, microorganisms transformed with the expression vector are provided. The host-vector system is not particularly limited, and for example, systems using Escherichia coli, actinomycetes, yeast, filamentous fungi, and fusion protein expression systems using these and other proteins can be used.

In addition, using the expression vector to transform microorganisms can be carried out according to methods routinely used in the art.

Then, the transformant is cultured in an appropriate medium, and the above-mentioned protein according to the present invention can be isolated from the culture. Therefore, according to another aspect of the present invention, there is provided a method for producing the novel protein according to the present invention. The culture of the transformant and its conditions may be substantially the same as those of the microorganisms used. In addition, methods for recovering the protein of interest after culturing the transformants can be those routinely used in this field.

Furthermore, according to a preferred aspect of the present invention, yeast cells capable of expressing endoglucanase and β-glucosidase encoded by the DNA sequence according to the present invention are provided. Examples of yeast cells in the present invention include microorganisms belonging to the genus Saccharomyces , Hansenula , or Pichia , such as Saccharomyces cerevisiae.

The host filamentous fungus in the present invention may belong to the genus Humicola , Aspergillus or Trichoderma , Fusarium , or Acremonium . And, as their preferred examples, can enumerate Humicola insolens ( Humicolainsolens ), Aspergillus niger ( Aspergillus niger) or Aspergillus oryzae ( Aspergillusoryzae ), or Trichodermaviride ( Trichodermaviride ), Fusarium oxysporum ( Fusariumoxysporum ), or fibrolytic Acremonium cellulolyticus.

By linking the endoglucanase and β-glucosidase genes according to the present invention with appropriate vectors and introducing them into Acremonium cellulolyticum, inhibiting their expression, or using homologous recombination to carry out genetic disruption of these genes to eliminate the defects Its function can inhibit the expression of specific endoglucanase and β-glucosidase. Gene disruption by homologous recombination can be carried out according to conventional methods, and the preparation of vectors for gene disruption and introduction of vectors into hosts are obvious to those skilled in the art.

Preparation of cellulase

The transformant thus obtained is cultured in an appropriate medium, and the above-mentioned protein of the present invention can be isolated from the culture. The culturing of the transformant and its conditions can be appropriately set according to the microorganisms used. In addition, the recovery and purification of the target protein from the culture solution can also be carried out according to conventional methods.

Cellulase preparation

According to another aspect of the present invention, there is provided a cellulase preparation comprising the above-mentioned protein (cellulase) according to the present invention. According to the cellulase preparation of the present invention, generally contained ingredients such as excipients (for example, lactose, sodium chloride, sorbitol, etc.), surfactants, Preservatives, etc. to manufacture. In addition, the cellulase preparation in the present invention can be prepared in any suitable shape, such as powder or liquid.

The use of cellulase

According to the present invention, it is believed that for biomass saccharification, saccharification can be significantly improved by treatment with the cellulase enzyme(s) or cellulase preparation of the present invention. Therefore, according to the present invention, there is provided an improved method of biomass saccharification, the method comprising the step of treating biomass with the cellulase(s) or cellulase preparation of the present invention. Examples of biomass that can be treated in the present invention include rice straw, bagasse, corn stover, fruit residues such as coconut nuts, waste wood, and materials obtained by subjecting these to appropriate pretreatment.

Furthermore, according to the present invention there is provided: a method for bringing about the clearing of fibers containing colored cellulose, the method comprising the step of treating fibers containing colored cellulose with a cellulase (group) or a preparation of cellulase . And, there is provided a method of bringing about a local change in color of fibers containing colored cellulose, that is, a method of imparting a stonewashed appearance to fibers containing colored cellulose. The method comprises the step of treating colored cellulose-containing fibers with an endoglucanase or cellulase preparation of the invention.

Furthermore, according to the present invention, it is considered that the drainage of paper or pulp can be significantly improved without accompanying significant reduction in strength by treatment with the endoglucanase according to the present invention. Therefore, according to the present invention, there is provided a method for improving the drainage of pulp, the method comprising the step of treating paper or pulp with the preparation of endoglucanase or cellulase of the present invention. Examples of pulp that can be treated in the present invention include waste paper pulp, recycled cardboard or pulp, kraft pulp, sulfurous acid pulp, heat-treated pulp, and other high-yield pulp.

Furthermore, by applying the endoglucanase according to the present invention to animal feed, the digestibility of glucan in the feed can be improved. Therefore, according to the present invention, there is provided a method for improving the digestibility of animal feed, the method comprising the step of treating animal feed with the endoglucanase or cellulase preparation of the present invention.

Example

Although the present invention will be specifically described by way of examples, the present invention is not limited to the following examples unless the gist is exceeded.

"Example 1: Cloning of ACC3 Gene"

(1-1) Isolation of genomic DNA

Acremonium cellulolyticus (Acremonium cellulolyticus) ACCP-5-1 strain was cultured in (s) medium (2% broth, 0.5% yeast extract, and 2% glucose) at 32°C for 2 days, and the bacteria were recovered by centrifugation. body. Genomic DNA was isolated from the obtained bacterial cells according to the method of Horiuchi et al. (H. Horiuchi et . al ., J. Bacteriol., 170, 272-278, (1988)).

(1-2) Acquisition of ACC3 gene fragment

The following primers were prepared based on the sequences of known endoglucanases classified in Glycoside Hydrolase family 5 (Glycoside Hydrolase family 5).

ACC3-F: GGGCGTCTGTRTTYGARTGT (SEQ ID NO::19)

ACC3-R: AAAATGTAGTCTCCCCCACCA (SEQ ID NO::20)

ACC3-F and ACC3-R were used as primers, and PCR was performed using genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented according to the following procedure: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; but the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified 1 kbp DNA fragment was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA Cloning Kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC3-partial.

Sequencing of the insert DNA fragment cloned into the plasmid TOPO-pACC3-partial was performed using BigDyeTerminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) and ABIPRISM GeneticAnalyzer (manufactured by Applied Biosystems) according to the attached protocol. As a result, the obtained nucleotide sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology. The result showed that it was 74% different from the endoglucanase EG1 (Q8WZD7) derived from Talaromycesemersonii . % identity, thus it was judged that the DNA fragment was a part of the endoglucanase (glycoside hydrolase family 5) gene.

(1-3) Obtain the full length of ACC3 gene by reverse PCR method

The inverse PCR method was carried out according to the method of Triglia et al. (TTriglia et . al ., Nucleic Acids Research, 16, 8186, (1988)). Genomic DNA of Acremonium cellulolyticum was digested overnight with Sal I, and circular DNA was prepared using MightyMix (manufactured by TaKaRaBio). Using this circular DNA as a template, PCR was performed with the following sequence contained in the ACC3 gene fragment to obtain the 5' upstream region and the 3' downstream region of the ACC3 gene.

ACC3-inv-F: ACTTCCAGACTTTCTGGTCC (SEQ ID NO::21)

ACC3-inv-R: AGGCCGAGAGTAAGTATCTC (SEQ ID NO::22)

For the above-mentioned 5' upstream region and 3' downstream region, the method described in Example 1-2 was used for analysis, and the full-length nucleotide sequence of the ACC3 gene was determined.

Based on the base sequence obtained by the inverse PCR method, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC3 gene.

pACC3-F: GAAGGATGGTAGATTGTCCG (SEQ ID NO::23)

pACC3-R: ACCGAGAAGGATTTCTCGCA (SEQ ID NO::24)

The amplified DNA was inserted into the pCR2.1-TOPO plasmid vector using TOPOTA cloning kit (manufactured by Invitrogen), to obtain plasmid pACC3. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC3 to obtain Escherichia coli TOP10 strain/pACC3.

(1-4) Preparation of cDNA and intron analysis of ACC3 gene

Acremonium cellulolyticum ACCP-5-1 strain was cultured with cellulase induction medium at 32°C for 2 days, and the bacterial cells were recovered by centrifugation. After freezing the obtained bacteria with liquid nitrogen, they were ground with a mortar and a grinding rod. Total RNA was isolated from the ground bacterial cells using ISOGEN (Nippongene) according to the attached protocol. Further, mRNA was purified from the total RNA using the mRNA Purification Kit (Pharmacia) according to the attached protocol.

From the thus obtained mRNA, cDNA was synthesized using TimeSavercDNA Synthesis Kit (Pharmacia) according to the attached protocol. The following primers including a start codon and a stop codon were prepared from the ACC3 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC3 cDNA gene.

ACC3-N: ATGAAGACCAGCATCATTTCTATC (SEQ ID NO::25)

ACC3-C: TCATGGGAAATAACTTCTCCAGAAT (SEQ ID NO::26)

The above-mentioned ACC3 cDNA gene was analyzed using the method described in Example 1-2, and the position of the intron was determined by comparing with the pACC3 gene.

(1-5) Estimation of the amino acid sequence of ACC3

The endoglucanase ACC3 gene isolated from Acremonium cellulolytica by the above method is composed of 1302 bp bases shown in the 136-1437 positions of the base sequence described in SEQ ID NO:1. In addition, the ACC3 gene contains 5 introns shown in positions 233-291, 351-425, 579-631, 697-754, and 853-907 of the base sequence described in SEQ ID NO: 1 . The amino acid sequence of ACC3 predicted from the open reading frame (ORF) is shown in SEQ ID NO:2. Furthermore, the -27 to -1 amino acid residues of this ACC3 were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 2: Cloning of ACC5 Gene"

(2-1) Isolation of genomic DNA and mRNA and preparation of cDNA

According to the method described in Example 1-1, the genomic DNA of Acremonium cellulosus ACCP-5-1 strain was isolated and analyzed. In addition, according to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was prepared.

(2-2) Acquisition of ACC5 gene fragment

The following primers were prepared based on the N-terminal amino acid sequence and poly A base sequence of known endoglucanases classified in Glycoside Hydrolase Family 7.

ACC5-F: CAGCAGGCCCCCACCCCNGAYAAYYTNGC (SEQ ID NO::27)

ACC5-R: AATTCGCGGCCGCTAAAAAAAAA (SEQ ID NO::28)

PCR was performed using ACC5-F and ACC5-R as primers and cDNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented according to the following procedure: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; but the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified 1.5 kbp DNA fragment was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA Cloning Kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC5-partial.

The base sequence of the inserted DNA fragment cloned into the plasmid TOPO-pACC5-partial was analyzed, the resulting base sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology, and the result was similar to that of Aspergillus fumigatus The source endoglucanase (Q4WCM9) showed 60% identity, so it was judged that this DNA fragment was a part of the endoglucanase (glycoside hydrolase family 7) gene.

(2-3) Obtain the full length of ACC5 gene by reverse PCR method

According to the method described in Example 1-3, using the circular DNA digested with HindIII as a template, PCR was carried out through the following sequence contained in the ACC5 gene fragment, and the 5' upstream region and the 3' downstream region of the ACC5 gene were obtained .

ACC5-inv-F: ATCTCACCTGCAACCTACGA (SEQ ID NO::29)

ACC5-inv-R: CCTCTTCCGTTCCACATAAA (SEQ ID NO::30)

The nucleotide sequences of the above-mentioned 5' upstream region and 3' downstream region were analyzed, and the full-length nucleotide sequence of the ACC5 gene was determined.

Based on the base sequence obtained by the inverse PCR method, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC5 gene.

pACC5-F: ATTGCTCCGCATAGGTTCAA (SEQ ID NO::31)

pACC5-R: TTCAGAGTTAGTGCCTCCAG (SEQ ID NO::32)

The amplified DNA was inserted into pCR2.1-TOPO plasmid vector using TOPOTA cloning kit (manufactured by Invitrogen), to obtain plasmid pACC5. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC5 to obtain Escherichia coli TOP10 strain/pACC5.

(2-4) Intron analysis of ACC5 gene

The following primers containing a start codon and a stop codon were prepared from the ACC5 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC5 cDNA gene.

ACC5-N: ATGGCGACTAGACCATTGGCTTTTG (SEQ ID NO::33)

ACC5-C:CTAAAGGCACTGTGAATAGTACGGA (SEQ ID NO::34)

The base sequence of the above-mentioned ACC5 cDNA gene was analyzed, and compared with the pACC5 gene, the position of the intron was determined.

(2-5) Estimation of the amino acid sequence of ACC5

The endoglucanase ACC5 gene isolated from Acremonium cellulolyticum by the above method is composed of 1488 bp bases shown in 128-1615 positions of the base sequence described in SEQ ID NO:3. The amino acid sequence of ACC5 predicted from the open reading frame (ORF) is shown in SEQ ID NO:4. Furthermore, the -20 to -1 amino acid residues of this ACC5 were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 3: Cloning of ACC6 Gene"

(3-1) Isolation of genomic DNA and preparation of genomic library

According to the method described in Example 1-1, the genomic DNA of Acremonium cellulosus ACCP-5-1 strain was isolated and analyzed. Isolated genomic DNA was partially digested with Sau 3AI. This was ligated to the Bam HI arm of the phage vector dMBL3 Cloning Kit (manufactured by Stratagene) using Ligation Kit Ver.2 (manufactured by Takara Shuzo Co., Ltd.). After ethanol precipitation, this was dissolved in TE buffer. The ligation mixture was formed into phage particles using MaxPlaxλ packaging kit (manufactured by Epicenter Technologies), and infected with Escherichia coli XL1-blueMRA (P2) strain. A genomic DNA library composed of ^1.1X10 phages was obtained by this method.

(3-2) Acquisition of ACC6 gene fragment

Based on the sequences of known endoglucanases classified in Glycoside Hydrolase Family 5, the following primers were prepared.

ACC6-F: GTGAACATCGCCGGCTTYGAYTTYGG (SEQ ID NO::35)

ACC6-R: CCGTTCCACCGGGCRTARTTRTG (SEQ ID NO::36)

PCR was performed using ACC6-F and ACC6-R as primers and genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented as follows: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; however, the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified 300 bp DNA fragment was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC6-partial.

The nucleotide sequence of the inserted DNA fragment cloned in the plasmid TOPO-pACC6-partial was analyzed, the resulting nucleotide sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology. The result was similar to that of Trichodermaviride The source endoglucanase EG3 (Q7Z7X2) showed 61% identity, so it was determined that this DNA fragment was a part of the endoglucanase (glycoside hydrolase family 5) gene. Using the plasmid TOPO-pACC6-partial as a template, this DNA fragment was used for PCR amplification in the same manner as above, and the resulting PCR product was labeled with ECLDIRECT SYSTEM (manufactured by Amersham Pharmacia Biotech) as a probe.

(3-3) Screening by colony hybridization

The phage colonies made in Example 3-1 were transferred to the Hybond-N+ nylon transfer membrane (manufactured by Amersham Company), and after alkaline denaturation, the bacteriophage colonies were transferred with 5 times the concentration of SSC (SSC: 15mmol/L trisodium citrate, 150mmol/L NaCl) and dried to immobilize the DNA. Hybridization was performed as follows: After 1 hour of prehybridization (42° C.), HRP-labeled probe was added, and hybridization was performed for 4 hours (42° C.). Probes were washed with 6M urea and 0.5-fold concentration SSC added with 0.4% SDS twice, and twice with 2-fold concentration SSC.

After immersing the probe-washed nylon membrane in the detection solution for 1 minute, it was exposed to HYPERFILMECL manufactured by the same company, and one positive clone was obtained. DNA preparation from positive clones was performed according to the method of Maniatis et al. (J. Sambrook, E.F. Fritschand T. Maniatis, "Molecular Cloning", Cold Spring Harbor Laboratory Press. 1989), using LE392 as host Escherichia coli. First, LE392 was cultured overnight with LB-MM medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride, 10 mmol/L magnesium sulfate, 0.2% maltose). It was infected with a phage solution derived from a single colony, and cultured overnight in LB-MM medium. Sodium chloride was added therein to make it 1M, and chloroform was added to make it 0.8%, to promote the lysis of Escherichia coli. Bacterial residues were removed by centrifugation, and phage particles were recovered from the 10% PEG6000 precipitate. Phage particles were digested with proteinase K in the presence of SDS, treated with phenol, and precipitated with ethanol to recover phage DNA.

Southern blot analysis was performed on the DNA prepared as above using ECLDIRECT SYSTEM. As a result of hybridization using the PCR amplified fragment of Example 3-2 as a probe, the 2.9 kbp Xba I fragment showed a common hybridization pattern with chromosomal DNA. The Xba I fragment was cloned into pUC118 to obtain plasmid pUC-ACC6, and then the base sequence of the plasmid was analyzed.

(3-4) Acquisition of full-length ACC6 gene

Based on the nucleotide sequence obtained from pUC-ACC6, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC6 gene.

pACC6-F: CTCTGCATTGAATCCCGAGA (SEQ ID NO::37)

pACC6-R: GCAACGCTAAAGTGCTCATC (SEQ ID NO::38)

The amplified DNA was inserted into pCR2.1-TOPO plasmid vector using TOPOTA cloning kit (manufactured by Invitrogen), to obtain plasmid pACC6. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC6 to obtain Escherichia coli TOP10 strain/pACC6.

(3-5) Preparation of cDNA and intron analysis of ACC6 gene

According to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was produced. The following primers containing a start codon and a stop codon were prepared from the ACC6 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC6 cDNA gene.

ACC6-N: ATGACAATCATCTCCAAAATTCGGT (SEQ ID NO::39)

ACC6-C:TCAGGATTTCCACTTTGGAACGAA (SEQ ID NO::40)

The base sequence of the above-mentioned ACC6 cDNA gene was analyzed, and compared with the pACC6 gene, the position of the intron was determined.

(3-6) Deduction of the amino acid sequence of ACC6

The endoglucanase ACC6 gene isolated from Acremonium cellulolyticum by the above method is composed of 1430 bp bases shown in positions 169-1598 of the base sequence described in SEQ ID NO:5. In addition, the present ACC6 gene contains three introns shown in positions 254-309, 406-461, and 1372-1450 of the base sequence described in SEQ ID NO:5. The amino acid sequence of ACC6 predicted from the open reading frame (ORF) is shown in SEQ ID NO:6. Furthermore, the -21--1 amino acid residues of this ACC6 were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 4: Cloning of ACC7 Gene"

(4-1) Isolation of genomic DNA and preparation of genomic library

According to the method described in Example 3-1, a genomic DNA library of Acremonium cellulosus ACCP-5-1 strain was prepared.

(4-2) Acquisition of ACC7 gene fragment

Based on the sequences of known endoglucanases classified in Glycoside Hydrolase Family 5, the following primers were prepared.

ACC7-F: CACGCCATGATCGACCCNCAYAAYTAYG (SEQ ID NO::41)

ACC7-R: ACCAGGGGCCGGCNGYCCACCA (SEQ ID NO::42)

PCR was performed using ACC7-F and ACC7-R as primers and genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented as follows: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; however, the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified DNA fragment of 670 bp was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC7-partial.

The nucleotide sequence of the inserted DNA fragment cloned in the plasmid TOPO-pACC7-partial was analyzed, and the obtained nucleotide sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology, and the result was derived from Aspergillus fumigatus The endoglucanase (Q4WM09) showed 63% identity, so it was judged that this DNA fragment was a part of the endoglucanase (glycoside hydrolase family 5) gene. Using the plasmid TOPO-pACC7-partial as a template, this DNA fragment was used for PCR amplification in the same manner as above, and the resulting PCR product was labeled with ECLDIRECT SYSTEM (manufactured by Amersham Pharmacia Biotech) as a probe.

(4-3) Screening by colony hybridization

The genomic DNA library was screened according to the method described in Example 3-3, and as a result, one positive clone was obtained. Southern blot analysis was performed on the obtained positive clones. As a result, the 3.7 kbp Xba I fragment showed a hybridization pattern common to chromosomal DNA. The Xba I fragment was cloned into pUC118 to obtain plasmid pUC-ACC7, and then the base sequence of the plasmid was analyzed.

(4-4) Acquisition of full-length ACC7 gene

Based on the nucleotide sequence obtained from pUC-ACC7, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC7 gene.

pACC7-F: CAGTCAGTTGTGTAGACACG (SEQ ID NO::43)

pACC7-R: ACTCAGCTGGGTCTTCATAG (SEQ ID NO::44)

The amplified DNA was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA Cloning Kit (manufactured by Invitrogen) to obtain plasmid pACC7. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC7 to obtain Escherichia coli TOP10 strain/pACC7.

(4-5) Preparation of cDNA and intron analysis of ACC7 gene

According to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was prepared. The following primers containing a start codon and a stop codon were prepared from the ACC7 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC7 cDNA gene.

ACC7-N: ATGAGGTCTACATCAACATTTGTA (SEQ ID NO::45)

ACC7-C: CTAAGGGGTGTAGGCCTGCAGGAT (SEQ ID NO::46)

The base sequence of the above-mentioned ACC7 cDNA gene was analyzed and compared with the pACC7 gene to determine the position of the intron.

(4-6) Deduction of amino acid sequence of ACC7

The endoglucanase ACC7 gene isolated from Acremonium cellulolytica by the above method is composed of 1307 bp bases shown in the 70-1376 positions of the base sequence described in SEQ ID NO:7. In addition, the present ACC7 gene contains 2 introns shown in positions 451-500 and 765-830 of the base sequence described in SEQ ID NO: 7. The amino acid sequence of ACC7 predicted from the open reading frame (ORF) is shown in SEQ ID NO:8. Furthermore, the -20 to -1 amino acid residues of this ACC7 were deduced as the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 5: Cloning of ACC8 Gene"

(5-1) Isolation of genomic DNA and preparation of genomic library

According to the method described in Example 3-1, a genomic DNA library of Acremonium cellulosus ACCP-5-1 strain was prepared.

(5-2) Acquisition of ACC8 gene fragment

Based on the DNA sequence corresponding to the N-terminal and C-terminal amino acid sequences of endoglucanase III derived from Penicillium verruculosum, the following primers were prepared.

MSW-N: CAACAGAGTCTATGCGCTCAATACTCGAGCTACACCAGT (SEQ ID NO::47)

MSW-C: CTAATTGACAGCTGCAGACCAA (SEQ ID NO::48)

PCR was performed using MSW-N and MSW-C as primers and genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented as follows: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; however, the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified DNA fragment of 800 bp was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC8-partial.

The nucleotide sequence of the inserted DNA fragment cloned in the plasmid TOPO-pACC8-partial was analyzed, the resulting nucleotide sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology. The result was similar to that of Trichodermaviride The derived endoglucanase Cel12A (Q8NJY4) showed 60% identity, so it was judged that this DNA fragment was a part of the endoglucanase (glycoside hydrolase family 12) gene. Using the plasmid TOPO-pACC8-partial as a template, this DNA fragment was used for PCR amplification in the same manner as above, and the resulting PCR product was labeled with ECLDIRECT SYSTEM (manufactured by Amersham Pharmacia Biotech) as a probe.

(5-3) Screening by colony hybridization

The genomic DNA library was screened according to the method described in Example 3-3, and as a result, one positive clone was obtained. Southern blot analysis was performed on the obtained positive clones. As a result, the approximately 5 kbp Sal I fragment showed a common hybridization pattern with chromosomal DNA. The Sal I fragment was cloned into pUC118 to obtain the plasmid pUC-ACC8, and the base sequence of the plasmid was analyzed.

(5-4) Acquisition of full-length ACC8 gene

Based on the nucleotide sequence obtained from pUC-ACC8, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC8 gene.

pACC8-F: AAAGACCGCGTGTTAGGATC (SEQ ID NO::49)

pACC8-R: CGCGTAGGAAATAAGACACC (SEQ ID NO::50)

The amplified DNA was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) to obtain plasmid pACC8. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC8 to obtain Escherichia coli TOP10 strain/pACC8.

(5-5) Preparation of cDNA and intron analysis of ACC8 gene

According to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was produced. The following primers containing a start codon and a stop codon were prepared from the ACC8 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC8 cDNA gene.

ACC8-N: ATGAAGCTAACTTTTCTCCTGAAC (SEQ ID NO::51)

ACC8-C:CTAATTGACAGATGCAGACCAATG (SEQ ID NO::52)

The base sequence of the above-mentioned ACC8 cDNA gene was analyzed and compared with the pACC8 gene to determine the position of the intron.

(5-6) Deduction of the amino acid sequence of ACC8

The endoglucanase ACC8 gene isolated from Acremonium cellulolytica by the above method is composed of 834 bp bases shown in positions 141-974 of the base sequence described in SEQ ID NO:9. In addition, the present ACC8 gene contains 2 introns shown in positions 551-609 and 831-894 of the base sequence described in SEQ ID NO: 9. The amino acid sequence of ACC8 predicted from the open reading frame (ORF) is shown in SEQ ID NO:10. Furthermore, the -15--1 amino acid residues of this ACC8 were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 6: Cloning of ACC9 Gene"

(6-1) Isolation of genomic DNA and mRNA and preparation of cDNA

According to the method described in Example 1-1, the genomic DNA of Acremonium cellulosus ACCP-5-1 strain was isolated and analyzed. In addition, according to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was prepared.

(6-2) Acquisition of ACC9 gene fragment

Based on the sequences of known endoglucanases classified in glycoside hydrolase family 45, the following primers were prepared.

ACC9-F: CCGGCTGCGGCAARTGYTAYMA (SEQ ID NO::53)

ACC9-R:AGTACCACTGGTTCTGCACCTTRCANGTNSC (SEQ ID NO::54)

PCR was performed using ACC9-F and ACC9-R as primers and genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented as follows: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; however, the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified DNA fragment of 800 bp was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC9-partial.

The base sequence of the inserted DNA fragment cloned in the plasmid TOPO-pACC9-partial was analyzed, the resulting base sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology, and the result was similar to that of Trichodermaviride The source endoglucanase EGV (Q7Z7X0) showed 79% identity, so it was judged that this DNA fragment was a part of the endoglucanase (glycoside hydrolase family 45) gene.

(6-3) Obtain the full length of ACC9 gene by reverse PCR method

According to the method described in Examples 1-3, using the circular DNA digested with Sal I or Xba I as a template, PCR was carried out through the following sequence contained in the ACC9 gene fragment, and the 5' upstream region of the ACC9 gene and 3 'Downstream area.

ACC9-inv-F: CGAAGTGTTTGGTGACAACG (SEQ ID NO::55)

ACC9-inv-R: GTGGTAGCTGTATCCGTAGT (SEQ ID NO::56)

The nucleotide sequences of the above-mentioned 5' upstream region and 3' downstream region were analyzed to determine the full-length nucleotide sequence of the ACC9 gene.

Based on the nucleotide sequence obtained by the inverse PCR method, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC9 gene.

pACC9-F: TACATTCCGAAGGCACAGTT (SEQ ID NO::57)

pACC9-R:CTGAGCTGATTATTCCTGACC (SEQ ID NO::58)

The amplified DNA was inserted into pCR2.1-TOPO plasmid vector using TOPOTA cloning kit (manufactured by Invitrogen) to obtain plasmid pACC9. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC9 to obtain Escherichia coli TOP10 strain/pACC9.

(6-4) Intron Analysis of ACC9 Gene

The following primers including a start codon and a stop codon were prepared from the ACC9 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC9 cDNA gene.

ACC9-N: ATGAAGGCTTTCTATCTTTCTCTC (SEQ ID NO::59)

ACC9-C:TTAGGACGAGCTGACGCACTGGTA (SEQ ID NO::60)

The base sequence of the above-mentioned ACC9 cDNA gene was analyzed and compared with the pACC9 gene to determine the position of the intron.

(6-5) Estimation of the amino acid sequence of ACC9

The endoglucanase ACC9 gene isolated from Acremonium cellulolyticum by the above method is composed of 1117bp bases shown in the 114th to 1230th positions of the base sequence described in SEQ ID NO:11. In addition, the present ACC9 gene contains 183-232 and 299-357 introns of the base sequence described in SEQ ID NO: 11. The amino acid sequence of ACC9 predicted from the open reading frame (ORF) is shown in SEQ ID NO:12. Furthermore, the -16--1 amino acid residues of this ACC9 were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 7: Cloning of ACC10 Gene"

(7-1) Isolation of genomic DNA and mRNA and preparation of cDNA

According to the method described in Example 1-1, the genomic DNA of Acremonium cellulosus ACCP-5-1 strain was isolated and analyzed. In addition, according to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was prepared.

(7-2) Acquisition of ACC10 gene fragment

Based on the sequences of known endoglucanases classified in Glycoside Hydrolase Family 61 and the poly A base sequence, the following primers were prepared.

ACC10-F: GGTGTACGTGGGCACCAAYGGNMGNGG (SEQ ID NO::61)

ACC10-R: AATTCGCGGCCGCTAAAAAAAAA (SEQ ID NO::62)

PCR was performed using ACC10-F and ACC10-R as primers and cDNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented according to the following procedure: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; but the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified 300 bp DNA fragment was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pACC10-partial.

The nucleotide sequence of the inserted DNA fragment cloned in the plasmid TOPO-pACC10-partial was analyzed, the resulting nucleotide sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology. The result was derived from Aspergillus sterreus The endoglucanase EGIV (QODOT6) showed 65% identity, so it was judged that this DNA fragment was a part of the endoglucanase (glycoside hydrolase family 61) gene.

(7-3) Obtain the full length of ACC10 gene by reverse PCR method

According to the method described in Examples 1-3, using the circular DNA digested with HindIII as a template, PCR was performed on the following sequence contained in the ACC10 gene fragment to obtain the 5' upstream region and 3' downstream region of the ACC10 gene.

ACC10-inv-F: TTCTGCTACTGCGGTTGCTA (SEQ ID NO::63)

ACC10-inv-R: GAATAACGTAGGTCGACAAG (SEQ ID NO::64)

The nucleotide sequences of the above-mentioned 5' upstream region and 3' downstream region were analyzed, and the full-length nucleotide sequence of the ACC10 gene was determined.

Based on the nucleotide sequence obtained by the inverse PCR method, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the ACC10 gene.

pACC10-F: CGTTGACCGAAAGCCACTT (SEQ ID NO::65)

pACC10-R: TGGCCTAAAGCTAAATGATG (SEQ ID NO::66)

The amplified DNA was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA Cloning Kit (manufactured by Invitrogen) to obtain plasmid pACC10. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pACC10 to obtain Escherichia coli TOP10 strain/pACC10.

(7-4) Intron Analysis of ACC10 Gene

The following primers including a start codon and a stop codon were prepared from the ACC10 gene sequence, and PCR was performed using the cDNA as a template to amplify the ACC10 cDNA gene.

ACC10-N: ATGCCTTCTACTAAAGTCGCTGCCC (SEQ ID NO::67)

ACC10-C:TTAAAGGACAGTAGTGGTGATGACG (SEQ ID NO::68)

The base sequence of the above-mentioned ACC10cDNA gene was analyzed and compared with the pACC10 gene to determine the position of the intron.

(7-5) Deduction of the amino acid sequence of ACC10

The endoglucanase ACC10 gene isolated from Acremonium cellulolyticum by the above method is composed of 1020 bp bases shown in the 124th to 1143rd positions of the base sequence described in SEQ ID NO:13. In addition, the present ACC10 gene contains an intron shown at positions 225 to 275 of the base sequence described in SEQ ID NO: 13. The amino acid sequence of ACC10 predicted from the open reading frame (ORF) is shown in SEQ ID NO: 14. Furthermore, the -21 to -1 amino acid residues of this ACC10 were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 8: Cloning of BGLC Gene"

(8-1) Preparation of genomic DNA and cDNA

According to the method described in Example 1-1, the genomic DNA of Acremonium cellulosus ACCP-5-1 strain was isolated and analyzed. In addition, according to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was prepared.

(8-2) Acquisition of BGLC gene fragments

Based on the sequences of known β-glucosidases classified in glycoside hydrolase family 1, the following primers were prepared.

BGLC-F: CCTGGGTGACCCTGTACCAYTGGGAYYT (SEQ ID NO::69)

BGLC-R: TGGGCAGGAGCAGCCRWWYTCNGT (SEQ ID NO::70)

PCR was performed using BGLC-F and BGLC-R as primers and genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented according to the following procedure: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; but the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified 1.2 kbp DNA fragment was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pBGLC-partial.

The base sequence of the inserted DNA fragment cloned in the plasmid TOPO- pBGLC -partial was analyzed, and the resulting base sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology. β-glucosidase 1 (Q4WRG4) showed 69% identity, so it was judged that this DNA fragment was a part of β-glucosidase (glycoside hydrolase family 1) gene.

(8-3) Obtain the full length of BGLC gene by reverse PCR method

According to the method described in Examples 1-3, using the circular DNA digested with Xba I as a template, PCR was performed on the following sequence contained in the BGLC gene fragment, and the 5'upstream region and 3'downstream region of the BGLC gene were obtained .

BGLC-inv-F: GGAGTTCTTCTACATTTCCC (SEQ ID NO::71)

BGLC-inv-R:AACAAGGACGGCGTGTCAGT (SEQ ID NO::72)

The nucleotide sequences of the above-mentioned 5' upstream region and 3' downstream region were analyzed, and the full-length nucleotide sequence of the BGLC gene was determined.

Based on the base sequence obtained by the inverse PCR method, the following primers were prepared, and PCR was performed using genomic DNA as a template to amplify the BGLC gene.

pBGLC-F: CTCCGTCAAGTGCGAAGTAT (SEQ ID NO::73)

pBGLC-R: GGCTCGCTAATACTAACTGC (SEQ ID NO::74)

The amplified DNA was inserted into the pCR2.1-TOPO plasmid vector using TOPOTA cloning kit (manufactured by Invitrogen) to obtain plasmid pBGLC. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pBGLC to obtain Escherichia coli TOP10 strain/pBGLC.

(8-4) Intron analysis of BGLC gene

The following primers including a start codon and a stop codon were prepared from the BGLC gene sequence, and PCR was performed using cDNA as a template to amplify the BGLC cDNA gene.

BGLC-N: ATGGGCTCTACATCTCCTGCCCAA (SEQ ID NO::75)

BGLC-C:CTAGTTCCTCGGCTCTATGTATTT (SEQ ID NO::76)

The base sequence of the above-mentioned BGLCcDNA gene was analyzed and compared with the pBGLC gene to determine the position of the intron.

(8-5) Deduction of amino acid sequence of BGLC

The β-glucosidase BGLC gene isolated from Acremonium cellulolytica by the above method is composed of 1650 bp bases shown in the 238th to 1887th positions of the base sequence described in SEQ ID NO:15. In addition, the present BGLC gene contains 3 introns shown in positions 784-850, 1138-1205, and 1703-1756 of the base sequence described in SEQ ID NO: 15. The amino acid sequence of BGLC predicted from the open reading frame (ORF) is shown in SEQ ID NO: 16. Furthermore, the -28 to -1 amino acid residues of this BGLC were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

"Example 9: Cloning of BGLD Gene"

(9-1) Preparation of genomic DNA and cDNA

According to the method described in Example 1-1, the genomic DNA of Acremonium cellulosus ACCP-5-1 strain was isolated and analyzed. In addition, according to the method described in Examples 1-4, cDNA for understanding Acremonium cellulosus ACCP-5-1 strain was prepared.

(9-2) Acquisition of BGLD gene fragment

Based on the sequences of known β-glucosidases classified in glycoside hydrolase family 1, the following primers were prepared.

BGLD-F: CACCGCCGCCTACCARRTNGARGG (SEQ ID NO::77)

BGLD-R: TGGCGGTGTAGTGGTTCATGSCRWARWARTC (SEQ ID NO::78)

PCR was performed using BGLD-F and BGLD-R as primers and genomic DNA as a template. PCR was carried out using LAtaq polymerase (manufactured by TaKaRaBio). PCR was implemented as follows: 94°C for 30 seconds, annealing for 30 seconds, 72°C for 1 minute, 40 cycles; however, the annealing temperature was reduced from 63°C to 53°C in stages in the first 20 cycles, and the subsequent 20 cycles fixed at 53°C. The amplified 1 kbp DNA fragment was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid TOPO-pBGLD-partial.

The nucleotide sequence of the inserted DNA fragment cloned in the plasmid TOPO-pBGLD-partial was analyzed, the resulting nucleotide sequence was translated into an amino acid sequence, and the amino acid sequence was searched for homology. The result was similar to that of T. emersonii ( Talaromycesemersonii )-derived β-glucosidase 1 (Q8X214) showed 76% identity, so it was judged that this DNA fragment was a part of β-glucosidase (glycoside hydrolase family 1) gene.

(9-3) Obtain the full length of BGLD gene by reverse PCR method

According to the method described in Examples 1-3, using the circular DNA digested with Xho I as a template, PCR was performed on the following sequence contained in the BGLD gene fragment, and the 5'upstream region and 3'downstream region of the BGLD gene were obtained .

BGLD-inv-F: CGGTTTCAATATCGGTAAGC (SEQ ID NO::79)

BGLD-inv-R: GTGTCCAAAAGCTCTGGAATG (SEQ ID NO::80)

The nucleotide sequences of the above-mentioned 5' upstream region and 3' downstream region were analyzed, and the full-length nucleotide sequence of the BGLD gene was determined.

Based on the nucleotide sequence obtained by the inverse PCR method, the following primers were prepared, and the BGLD gene was amplified by PCR using genomic DNA as a template.

pBGLD-F: TTCTCTCACTTTCCCTTTCC (SEQ ID NO::81)

pBGLD-R: AATTGATGCTCCTGATGCGG (SEQ ID NO::82)

The amplified DNA was inserted into the pCR2.1-TOPO plasmid vector using the TOPOTA cloning kit (manufactured by Invitrogen) to obtain the plasmid pBGLD. Escherichia coli TOP10 strain (manufactured by Invitrogen) was transformed with the obtained plasmid pBGLD to obtain Escherichia coli TOP10 strain/pBGLD.

(9-4) Intron analysis of BGLD gene

The following primers including a start codon and a stop codon were prepared from the BGLD gene sequence, and PCR was performed using cDNA as a template to amplify the BGLD cDNA gene.

BGLD-N: ATGGGTAGCGTAACTAGTACCAAC (SEQ ID NO::83)

BGLD-C: CTACTCTTTCGAGATGTATTTGTT (SEQ ID NO::84)

The base sequence of the above-mentioned BGLD cDNA gene was analyzed, and compared with the pBGLD gene, the position of the intron was determined.

(9-5) Deduction of amino acid sequence of BGLD

The β-glucosidase BGLD gene isolated from Acremonium cellulolytica by the above method consists of 1700 bp bases shown in positions 66-1765 of the base sequence described in SEQ ID NO: 17. In addition, the present BGLD gene contains four introns shown in positions 149-211, 404-460, 934-988, and 1575-1626 of the base sequence described in SEQ ID NO: 17. The amino acid sequence of BGLD predicted from the open reading frame (ORF) is shown in SEQ ID NO: 18. Furthermore, the -33 to -1 amino acid residues of this BGLD were deduced to be the signal sequence by using the signal sequence prediction software SignalP3.0.

Industrial Applicability

The protein of the present invention can be used as a cellulase preparation, and can be suitably used for decomposing cellulosic substrates.

As mentioned above, the present invention has been described through specific aspects, and modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.

Each base sequence shown in the sequence of SEQ ID NO: 19-84 in the sequence listing is an artificially synthesized primer sequence. SEQ ID NO: 27 (18 and 27), SEQ ID NO: 41 (18), SEQ ID NO: 42 (14), SEQ ID NO: 54 (26 and 29), SEQ ID NO: 61 (22 and 25), SEQ ID NO The symbol "n" in : 70 (position 22) and SEQ ID NO: 77 (position 19) represents an arbitrary base.

Claims (24)

1. the DNA that the base sequence shown in SEQIDNO:13 is constituted.

2. the protein of the Sequence composition of 1～301 of the aminoacid sequence shown in SEQIDNO:14.

3. coding protein DNA described in claim 2.

4. the DNA of the Sequence composition of 124～1143 of the base sequence shown in SEQIDNO:13.

5. the DNA removing intron sequences from the DNA according to any one of claim 3-4 and obtain.

6. the DNA described in claim 5, wherein, intron sequences is the sequence of 225～275 of the base sequence shown in SEQIDNO:13.

7. the DNA removing the base sequence of coded signal sequence from the DNA according to any one of claim 3,4 and 6 and obtain.

8. the DNA removing the base sequence of coded signal sequence from the DNA described in claim 5 and obtain.

9. the DNA according to any one of claim 7-8, wherein, the base sequence of coded signal sequence is the sequence of 124～186 of the base sequence shown in SEQIDNO:13.

10. the polynucleotide that the base sequence of the protein described in coding claim 2 is constituted.

11. comprise the expression vector of DNA according to any one of claim 1,3～9.

12. the host cell converted with the expression vector described in claim 11.

13. the host cell described in claim 12, wherein, host cell is yeast or filamentous fungi.

14. the host cell described in claim 13, wherein, yeast belongs to Saccharomycodes (Saccharomyces), Hansenula (Hansenula) or pichia (Pichia).

15. the host cell described in claim 14, wherein, yeast is saccharomyces cerevisiae (Saccharomycescerevisiae).

16. the host cell described in claim 13, wherein, filamentous fungi belongs to Humicola (Humicola), aspergillus (Aspergillus), trichoderma (Trichoderma), Fusarium (Fusarium) or branch acremonium and belongs to (Acremonium).

17. the host cell described in claim 16, wherein, filamentous fungi is to solve cellulose branch top spore (Acremoniumcellulolyticus), Humicola insolens (Humicolainsolens), aspergillus niger (Aspergillusniger) or aspergillus oryzae (Aspergillusoryzae), Trichoderma viride (Trichodermaviride) or pinch outs (Fusariumoxysporum).

18. the manufacture method of the protein described in claim 2, it comprises the steps: to cultivate the host cell according to any one of claim 12～17, and collects the protein described in claim 2 from this host and/or its culture.

19. Cellulase preparation, it comprises the protein described in claim 2.

20. Biomass carries out a processing method for saccharifying, the method comprises the steps: to make to contact with the protein described in claim 2 or the Cellulase preparation described in claim 19 containing cellulosic Biomass.

21. containing a processing method for cellulosic fiber, the method comprises the steps: to make to contact with the protein described in claim 2 or the Cellulase preparation described in claim 19 containing cellulosic fiber.

22. the de-inking method of a waste paper, it is characterised in that processing in the step that waste paper carries out deinking with deinking agent, use the protein described in claim 2 or the Cellulase preparation described in claim 19.

23. the method improving the drainability of paper or paper pulp, the method comprises the steps: to process the protein described in paper or paper pulp claim 2 or the Cellulase preparation described in claim 19.

24. the method improving the digestibility of animal feed, the method includes: the protein described in animal feed claim 2 or the Cellulase preparation described in claim 19 carry out the step processed.